Previous studies in my laboratory have indicated multiple symptoms of accelerated aging in mice deleted for Ku80, which is known to act in a variety of chromatin-related cellular processes, most notably in the repair of double-strand breaks through non-homologous end joining. The long-term objective of Project 4 is to further investigate the role of Ku80 in longevity assurance, based on its genome maintenance function and its potential genetic interactions with a variety of recently emerged potential cellular defense systems. The proposed studies are in keeping with the central theme of the program project: i.e., oxidative stress-induced DNA damage as a major driving force of aging-related cellular degeneration and death. This long-term objective will be pursued along four specific aims, worked out in close collaboration with the other projects of this PPG, and extensively using its core facilities. For Aim 1, we will analyze ku80 -/- mice crossed into various backgrounds that are compromised for chromatin metabolism (Rad54, DNA-PKcs, Ku70) or cell cycle response, (p53 and p27/Kipl)(Collaboration with projects 1-3, Core B and Core C). For Aim 2 we will screen the recently emerged candidate longevity assurance genes Foxo3a, Sir2alpha, Sir2L2 and Sir2L3, and we will screen a gene important for a signal transduction pathway that regulates oxidative stress responses, p66/shc. We will generate and analyze genetically altered cells for these genes and investigate the potential influence of the gene products on a multitude of DNA metabolism pathways, using cell culture systems and a genotoxic screen recently developed in the laboratory (Collaboration with projects 1-3 and core C). For Aim 3, we will test the hypothesis that these genes or altered genes tested in aim 2, alone or in combination, can be used to ameliorate the accelerated aging symptoms in the ku80 -/- cells, and in the accelerated aging mutants discovered by our collaborators of project 1. The results from aim 2 will determine how we proceed for aim 3. This part of the project provides extensive synergy with projects 1 and 3. For Aim 4, we will develop genetic tools to facilitate analysis of mice and cells. This consists of two parts: (1) development of tools that enable over-expression of multiple anti-oxidant defense genes at the same locus (which will avoid the extensive breeding that is now necessary to obtain such mice); and (2) development of a mouse model harboring one or more senescence reporters. These two mouse-modeling tools will be developed in close collaboration with projects 1 and 3, respectively.